3,5-dimethoxybenzyl carbonate esters



United States Patent 3,467,690 3,5-DIMETHOXYBENZYL CARBONATE ESTERSJames W. Chamberlin, Indianapolis, Ind., assignor to Eli Lilly andCompany, Indianapolis, Ind., a corporation of Indiana No Drawing. FiledMay 12, 1965, Ser. No. 455,307 Int. Cl. C07c 69/00, 79/00, 99/00 US. Cl.260--463 3 Claims ABSTRACT OF THE DISCLOSURE 3,5-dimethoxybenzyloxycarbonate esters, azides, and halides, useful inpeptide synthesis to protect the amino group of the N-terminal aminoacid, from which the protecting group is quantitatively cleaved byirradiation with ultraviolet light.

This invention relates to certain compounds useful in the art ofpreparing peptides.

Since peptides are important biological substances, and their isolationfrom biological systems in a pure state is difficult, it is necessary toprepare these materials by synthetic chemical methods. These methodsinvolve as a fundamental step the coupling of two or more amino acids ina manner to form an amido linkage between the molecules:

0 O 4:115 m. ill-sali- Since amino acids are at least bifunctional, itis also necessary for the chemist to render inactive all functionalitiesin a given amino acid which are not directly used in the amido couplingprocess prior to the aforesaid coupling process. If reactivefuntionalities are allowed to remain, yields will be lower andpurifications made 'difficult because of the presence of large amountsof unwanted byproducts from the interaction of these functionalities.Several methods are well known by the chemist for render-ing inactivethe functionalities of simple amino acids with protecting groups in sucha manner that only the desired functional group is available to reactwhen the amido linkage is formed. It is necessary for the so-calledprotecting group to be readily attached to the amino acid before amideformation and to be readily removed from the resulting peptide, aftercoupling, without simultaneous rupture of the newly formed amidelinkage. Two types of protecting groups are necessary in peptidesynthesis: the C-terminal protecting groups, those groups which renderthe acid portion of the amino acid inactive, as for example, alcoholderivatives, to prepare esters; amine derivatives, to prepare amides;and the like; and the N-terminal protecting groups, those groups whichrender the amine portion unreactive, such as benzyloxycarbonyl, trityl,allyloxy and the like. It is with the latter type of protecting groupthat this invention is concerned. Typical of the N-terminal protectinggroups is the benzyloxycarbonyl group. This group forms, with nitrogenatom, a carbamate linkage and is readily attached to the amino acid bymethods well known in the art. The benzyloxycarbonyl-protected aminoacid can then be used to prepare a dipeptide, that is, a molecule madeup of two amino acid moities. After the protected amino acid has beenreacted to form the desired peptide linkage, it becomes necessary toremove the benzyloxycarbonyl group. The methods available to the chemisthave classically been hydrogenolysis or hydrolysis. These reactionsideally must favor the cleavage of the carbamate bond which forms theprotecting group and to leave untouched 3,467,690 Patented Sept. 16,1969 the peptide linkage. Most methods are only moderately successful inthis regard. The reactions involved are equilibrium reactions andusually cause some cleavage of the amide bond.

It is an object of this invention to provide a protected amino acid inwhich the amine-protecting group can be easily prepared, attached to theamino acid, and then removed therefrom nearly quantitatively by actinicradiation, preferably ultraviolet light. It is also an object of thisinvention to provide a protected amino acid in which theamino-protecting group can be removed without the addition of acid orbase. Other objects will be apparent from the following description.

In fulfiillment of the above and other objects, this invention employsnovel compounds represented by the following formula:

CH O

i CH2OCR1 CHaO wherein R is halo, azido, or p-nitrophenoxy, saidcompounds being inactive intermediates for the preparation of N-terminalprotected amino acids of the formula CHaO wherein R when taken alone, ishydrogen or C -C alkyl;

R when taken alone, is hydrogen, C C alkyl, hydroXy-substituted C -Calkyl, carboxy-substituted C -C alkyl, lower alkyl mercapto-substitutedC -C alkyl, guanidino-substituted C -C alkyl, guanidinooxy-substituted C-C alkyl, imidazolymethyl, indolylmethyl, thineyl, furyl, phenyl, orbenzyl; and

R and R when taken together with the atoms to which they are attached,are piperidine or pyrrolidine.

C -C alkyl as used herein includes methyl, ethyl, ngropyl, isopropyl,n-butyl, isobutyl, sec.-butyl, and tert.- utyl.

Lower alkyl includes methyl, ethyl, n-propyl, and isopropyl.

Lower alkoxy includes methoxy, ethoxy, n-propoxy, and isopropoxy.

Halo includes fluoro, chloro, and bromo.

Lower alkylmercapto includes methylthio, ethylthio, n-propylthio, andisopropylthio.

While the compounds of the present invention have been defined ascontaining certain well-recognized organic radicals-4n particular Rwhich may include phenyl, benzyl, and certain heterocyclic radicals-itwill be recognized that such radicals may bear one or more substituentswithout departing in any way from the spirit of the invention andwithout any resultant alteration in the properties of the compoundswhich would set them apart from the invention or take them outside itsscope. Compounds bearing such substituents are accordingly to beconsidered as equivalents of the defined compounds and as lying withinthe scope of the invention. Among such substituent atoms and radicalsare phenyl, halo, lower alkyl, perhalolower alkyl, lower alkoxy, loweralkylmercapto, cyano, acetyl, acetamido, hydroxy, hydroxymethyl,fi-hydroxyethyl, and the like.

Illustrative compounds coming within the scope of this invention includethe following:

N-3,5-dimethoxybenzoxycarbonyl L-tyr-osineN-3,S-dimethoxybenzoxycarbonyl alanine N-3,S-dimethoxybenzoxycarbonylphenylalanine N-3,S-dimethoxybenzoxycarbonyl a-amino-n-butyric acidN-3,S-dimethoxybenzoxycarbonyl D-valine N-3,5-dimethoxybenzoxycarbonyl2,4-dichlorophenylalanine N-3,S-dimethoxybenzoxycarbonyl a-aminoadipicacid N-3,S-dimethoxybenzoxycarbonyl arginineN-3,S-dimethoxybenzoxycarbonyl L-proline N-3,5-dimethoxybenzoxycarbonylS-ethylcysteine N-3,5-dimethoxybenzoxycarbonyl2,6-dimethoxyphenylglycine N-3,5-dimethoxybenzoxycarbonyl aspartic acidN-3,S-dimethoxybenzoxycarbonyl canavanine N-3,S-dimethoxybenzoxycarbonyl3,5-diiodotyrosine N-3,5-dimethoxybenzoxycarbonyl ethionineN-3,5-dimethoxybenzoxycarbonyl glutamic acidN-3,S-dimethoxybenzoxycarbonyl histidine N-3,S-dimethoxybenzoxycarbonylhomoserine N-3,S-dimethoxybenzoxycarbonyl isoleucineN-3,5-dimethoxybenzoxycarbonyl tert.-leucineN-3,S-dimethoxybenzoxycarbonyl isovaline N-3,5-dimethoxy-benzoxycarbonylnorleucine N-3,S-dimethoxybenzoxycarbonyl norvalineN-3,S-dimethoxybenzoxycarbonyl methionine N-3,S-dimethoxybenzoxycarbonyll-methylhistidine N-3,5-dimethoxybenzoxycarbonyl pipecolic acidN-3,5-dimethoxybenzoxycarbonyl sarcosine N-3,S-dimethoxybenzoxycarbonylserine N-3,S-dimethoxybenzoxycarbonyl threonineN-3,S-dimethoxybenzoxycarbonyl tryptophan Compounds of the presentinvention can be prepared in the following manner. 3,5-dimethoxybenzylalcohol is prepared from the known 3,5-dimethoxybenzoic acid by any ofthe usual reductive procedures, such as catalytic hydrogenation orreaction with lithium aluminum hydride. The resulting carbinol istreated with phosgene in the presence of an acid scavenger, as forexample, dimethyl aniline, pyridine, or the like, to prepare the3,5-dimethoxybenzyl chlorocarbonate. The chlorocarbonate is reacteddirectly with the amino acid to obtain the protected amino acid, or itcan be converted to an azide by reaction with hydrazine and subsequentdiazotization with an inorganic nitrite to produce the azide, which isreacted with the amino acid. The azide can also be prepared by treatmentof the chlorocarbonate with an inorganic azide, as for example, sodiumazide. As a further alternative, the 3,5-dimethoxybenzyl alcohol can beconverted to the p-nitrophenyl carbonate by treatment with p-nitrophenylchlorocarbonate in the presence of an acid scavenger, as for example,pyridine, N,N-dimethylaniline, or the like. The p-nitrophenyl carbonatederivative can then be reacted with the amino acid using aqueous sodiumhydroxide solution as a condensing agent.

Cleavage of the protective group from the protected amino acid orpeptide can be carried out with an apparatus equipped for the emissionof ultraviolet light. A typical apparatus for such as procedure is theHanovia 654A-36 high-pressure mercury lamp. Compounds to be irradiatedshould be dissolved in an aqueous medium, optionally with an organicsolvent added for improved solubility. A typical solvent for theirradiation of most peptides is a 1:1 dioxane-water mixture.

3,5-dimethoxybenzyl alcohol for use in the present invention wasprepared in the following manner:

To a mixture of 22.8 g. (0.601 mole) of lithium aluminum hydride and 300ml. of tetrahydrofuran was added with stirring a solution of 110 g.(0.605 mole) of 3,5- dimethoxybenzoic acid in 800 ml. oftetrahydrofuran. The resulting mixture was refluxed for 20 hours andthen stirred for an additional 3 hours. The excess lithium aluminumhydride was decomposed with saturated sodium sulfate solution, themixture filtered, and the filtrate dried with sodium sulfate-magnesiumsulfate. Evaporation of the solvent yielded 75.6 g. (74 percent) of3,5-dimethoxy- 4 benzyl alcohol; M.P. 48-50 C.; A 220 ma (6 7410), 280mu (1760).

EXAMPLE 1 3,5-dimethoxybenzyl p-nitrophenyl carbonate To an ice-coldsolution of 28.0 g. (0.167 mole) of 3,5- dimethoxybenzyl alcohol and5.90 g. (0.150 mole) of pyridine in ml. of acetone were added withstirring 30.4 g. (0.150 mole) of p-nitrophenyl chloroformate.Immediately after the last portion had been added, a solid product beganto precipitate. The resulting slurry was added to 700 ml. of water, thesolid collected by filtration, washed several times with water, andrecrystallized from methanol (2500 ml.). There were obtained 30.7 g. (61percent) of 3,5-dimethoxybenzyl p-nitrophenyl carbonate, M.P. 1l4115 C.

Analysis.Calcd. for C H NO C, 57.65; H, 4.54; N, 4.20. Found: C, 57.91;H, 4.77; N, 3.95.

EXAMPLE 2 N-3,S-dimethoxybenzyloxycarbonyl-D-phenylglycine A mixture of3.66 g. (0.024 mole) of D-phenylglycine, 10.0 g. (0.030 mole) of3,5-dimethoxybenzyloxy p-nitrophenyl carbonate, 24.0 ml. of 2 N sodiumhydroxide solution, and 48 ml. of tetrahydrofuran was stirred at roomtemperature for 21 hours. The tetrahydrofuran was evaporated underreduced pressure. The precipitated solid was collected by filtration andwashed several times with aqueous 1 M sodium bicarbonate solution. Thecombined filtrates were adjusted to pH 5.8 with 1 N hydrochloric acid,and the resulting mixture was extracted three times with ether to removep-nitrophenol and any unreacted 3,5-dimethoxybenzyloxy p-nitrophenylcarbonate. The aqueous phase was then over-layered with ether andacidified to pH 2.0. The ether layer was removed and the aqueous layerwas extracted twice more with ether. The ether extracts were combined,washed twice with water, once with saturated sodium chloride solution,and dried with sodium sulfate. Evaporation of the solvent andcrystallization of the residue from methanol-water afforded 6.40 g. (77percent) of 3,5-dimethoxybenzyloxycarbonyl-D-phenylglycine, M.P. 132.5C. The analytical sample melted at 132.5133.5 C., [a] 100 (C. 1.05,chloroform), pK' 5.5 (66 percent aqueous dimethylformamide), k 280 my.(:2 1930).

Analysis.-Calcd. for C H NO C, 62.60; H, 5.54; N, 4.06. Found: C, 62.89;H, 5.57; N, 4.04.

Table 1 lists the physical constants of four additional amino acidsprepared by the above procedure.

for GizHrslglo z C, 53.53; H, 5.62. Found: C,

DL-methionine Yield, 68%; M.P. 86-88 0.; neutral equivalent calculated;343; found: 337; pKn (66% dimethylformamide) 5.95; analysis calulatedfor C HmNO S: C, 52.46; H, 6.11. Found: C, 52.68; H, 6.18.

L-serine Yield 65%; M.P. 4456 0.; [a]n +15.0 (C, 1.02) neutralequivalent calculated; 299; found: 296; pK (66% dimethyliormamide) 5.85;analysis calculated for C|3H|7NO72 C, 52.17; H, 5.73. Found: C, 51.91;H, 5.97.

Yield, 74%; M.P. 120.5-121. 5 0.; [a]D +11.7 (OHClg, O, 1.02);neutralization equivalent: calculated, 475; found, 480; pK 6.05 (66%DMF); analysis: calculated for CztHaoNzOgZ C, 60.75; H, 6.37. Found: C,60.98; H, 6.51.

e-Carbobenzoxy-L- lysine.

EXAMPLE 3 N-3,S-dimethoxybenzyloxycarbonyl-D-phenylglycylglycine Asolution of 1.38 g. (0.004 mole) of3,5-dimethoxybenzyloxycarbonyl-D-phenylglycine in 20 ml. oftetrahydrofuran was cooled in a salt-ice bath and 0.56 ml. oftriethylamine was added, followed by 0.52 ml. of isobutyl chloroformate.The mixture was stirred for minutes. A cold solution of 0.300 g. (0.004mole) of glycine and 0.56 ml. of triethylamine in m1. of 1:1water-tetrahydrofuran was then added dropwise, and the mixture wasstirred for one hour in the cold and one hour at room temperature. Thetetrahydrofuran was removed under reduced pressure. The residue wasdiluted with 20 ml. of water and washed once with 20 ml. of ethylacetate. The aqueous phase was separated, diluted with 40 ml. of water,over-layered with 40 ml. of ethyl acetate, and the pH adjusted to 2.5with 10 percent hydrochloric acid. The aqueous phase was extracted oncemore with 40 ml. of ethyl acetate. The ethyl acetate extracts werecombined, washed once with water and dried with anhydrous sodiumsulfate. The solid residue obtained upon evaporation of the solvent wasslurried with ether and filtered to give 1.16 g. (72 percent) of3,5-dimethoxybenzyloxycarbonyl-D-phenylglycylg1ycine, M.P. 156-157 C.The analytical sample was obtained by recrystallization frommethanol-water, M.P. 156.5157.5 C. [a] 60.3 (c.=0.823, ethanol), pK 5.9(66% aqueous dimethylformamide).

Analysis.-Calcd. for C20H22N207: C, 59.69; H, 5.51; N, 6.96. Found: C,59.42; H, 5.68; N, 6.69.

General procedure and apparatus for irradiation The irradiations werecarried out with a Hanovia 654A-36 high-pressure mercury lamp housed ina Watercooled quartz immersion well and fitted with a Vycor glass(Corning No. 7910) filter sleeve. The compound to be irradiated wasdissolved in one liter of 1:1 dioxane-water. Nitrogen was passed throughthe solution during the course of the reaction. An irradiation time of1.5 hours was employed for the preparative experiments. The progress ofthe reactions was followed by paper chromatography (ninhydrin). In allcases studied, no free amino acid was detected by this method in aliquotsamples maintained in the dark during the irradiation period. Thedipeptide and the amino acids were isolated by evaporating the solutionto dryness, treating the residue with a minimum volume of acetone, andcollecting the product by filtration.

EXAMPLE 4 Irradiation of N-3,S-dimethoxybenzyloxyglycine A solution 'of1.345 g. (0.005 mole) of 3,5-dimethoxybenzyloxycarbonylglycine in oneliter of 1:1 dioxanewater was irradiated by the general procedure abovefor 1.5 hours. The clear solution was evaporated to dryness, and theresidue was treated with a small amount of acetone to crystallize theresulting glycine. Yield: 0.318 g. (85%), pK' 3.85, 10.35 (66% aqueousdimethylformamide). Neutral equiv.

Analysis.--Calcd. for C H NO 75.1. Found: 78.2.

The acetone filtrate from the filtration of the crystallized glycine wasevaporated to dryness. The residue was taken up in ethyl acetate andpassed through a column of Woelm neutral alumina (activity III, 25 g.)Elution with ethyl acetate provided 0.734 g. of yellowish oil thatcrystallized on standing, M.P. 34-43 C. The infrared spectrum of thismaterial was identical with that of 3,5- dimethoxybenzyl alcohol.Recrystallization from Skellysolve B afforded 0.235 g. of the alcohol,M.P. 43-46 C., and a second crop of 0.088 g., M.P. 40-45 C. The totalyield of recrystallized 3,5-dimethoxybenzy1 alcohol amounted to 38percent.

Table 2 lists the yield of purified amino acid recovered uponirradiation of the 3,5-dimethoxybenzyloxy carbonyl derivative.

TABLE 2 Amino acid: Yield, percent DL-methionine D-phenylglycine 66L-serine 72 e-CarbobenzoXy-L-lysine (of the e-carbobenzoxy protectedamino acid) 62 EXAMPLE 5 wherein R is a member of the group consistingof fiuoro, chloro, bromo, and p-nitrophenoxy.

2. 3,5-dimethoxybenzyloxycarbonyl chloride.

3. 3,5-dimethoxybenzyl p-nitrophenyl carbonate.

No references cited.

CHARLES B. PARKER, Primary Examiner L. C. MARUZO, Assistant ExaminerU.S. Cl. X.R.

